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Minibrain kinase enhances synaptojanin activity to facilitate endocytosis during synaptic activity
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Minibrain kinase enhances synaptojanin activity to facilitate endocytosis during synaptic activity
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Content
MINIBRAIN KINASE ENHANCES SYNAPTOJANIN
ACTIVITY TO FACILITATE ENDOCYTOSIS DURING
SYNAPTIC ACTIVITY
by
Chun-Kan Chen
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY & MOLECULAR BIOLOGY)
August 2013
Copyright 2013 Chun-Kan Chen
ii
ACKNOWLEDGEMENTS
First of all, I’d like to thank my mentor, Dr. Karen T. Chang (University of Southern
California, CA, USA), who led me to become an independent researcher and gave me
valuable advice that guided me to finish this project. I would also like to send my thanks
to Catherine Bregere, who conducted preliminary experiments that inspired that latter
proportion of this project. In addition, I want to thank my lab mates, Jillian Shaw, Joo
Yeun Lee, and Dr. Derek Sieburth (University of Southern California, CA, USA) and his
lab members, who provided helpful comments to move the project forward during lab
meetings. I am also grateful to Dr. Wange Lu (University of Southern California, CA,
USA), who let me join his lab meeting to sharpen my critical thinking.
I like to acknowledge Dr. Dion Dickman (University of Southern California, CA,
USA) for the collaboration and his critical comments on this project, and Jeremy Paluch,
an undergraduate student form Dr. Dickman’s lab, who conducted all the
electrophysiology experiments. I also want to thank Dr. Hugo Bellen (Baylor, TX, USA)
for his generous sharing of UAS-PLCδ1-PH-GFP flies and multiple antibodies used in
this thesis, including the synaptojanin antibody that we renamed here to p-Synj for
clarification purposes.
iii
Last but not least, I would like to send my special thanks to Christine Juang and my
parents and brother, who provided both instrumental and emotional support to assist me
in completing this MS degree. I am truly grateful for all the support. Thank You.
iv
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ............................................................................................. ii
TABLE OF ABBREVIATIONS ..................................................................................... vii
ABSTRACT .................................................................................................................... viii
CHAPTER 1: INTRODUCTION .................................................................................... 1
1.1 Synaptic endocytosis ............................................................................................. 1
1.2 Study endocytosis in neuromuscular junctions of Drosophila melanogaster ....... 1
1.2.1 Drosophila melanogaster ........................................................................... 1
1.2.2 Drosophila neuromuscular junction ........................................................... 2
1.3 Endocytosis regulation .......................................................................................... 3
1.3.1 Synaptic kinases ......................................................................................... 3
1.3.2 Minibrain kinase ........................................................................................ 4
1.3.3 Synaptojanin .............................................................................................. 5
1.4 Goals and significance of the research .................................................................. 6
CHAPTER 2: EXPERIMENTAL PROCEDURES ....................................................... 8
2.1 Fly Stocks and antibody generation ...................................................................... 8
2.2 Immunochemistry ................................................................................................. 8
2.3 Electrophysiology and FM1-43 dye labeling ........................................................ 9
2.4 Western blotting ...................................................................................................11
2.5 Immunoprecipitation ............................................................................................11
2.6 Phosphoprotein separation .................................................................................. 12
2.7 Protein purification ............................................................................................. 12
2.8 In vitro dephosphorylation and re-phosphrylation of Synj ................................. 13
2.9 Phosphatidylinositol 5’-phosphatase activity in vitro ......................................... 14
2.10 In vitro Synj interaction with endocytotic proteins ........................................... 15
2.11 Synapse activity stimulation by high K
+
........................................................... 15
CHAPTER 3: RESULTS ................................................................................................ 17
3.1 Mnb is enriched in the presynaptic terminals of Drosophila NMJ ..................... 17
3.2 Mnb is required for synaptic growth ................................................................... 19
3.3 Mnb is required for rapid synaptic vesicle recycling .......................................... 22
3.4 Synaptojanin is a substrate for Mnb.................................................................... 26
3.5 Mnb regulates interaction of endocytic proteins and enhances Synj
5’-phosphoinositol phosphatase activity ............................................................ 31
v
3.6 Mnb phosphorylates and enhances synaptojanin during synaptic activity ......... 37
3.7 Overexpression of Synaptojanin restores synaptic bouton size and endocytic
function in mnb
1
mutants .................................................................................... 41
CHAPTER 4: DISCUSSIONS AND CONCLUSIONS ............................................... 48
4.1 Mnb-mediated phosphorylation enhances Synj phosphatase activity ................ 48
4.2 Mnb-mediated phosphorylation of Synj regulates proper endocytic protein
interactions and recruitments during synaptic activity ...................................... 48
4.3 Mnb regulates synaptic morphology through Synj phosphorylation and other
pathways ............................................................................................................ 50
4.4 Future directions ................................................................................................. 53
REFERENCES ................................................................................................................ 54
vi
LIST OF FIGURES
Fig. 3.1-1 Mnb is found in the presynaptic terminals of Drosophila NMJ ....................... 20
Fig. 3.1-2 Mnb isoforms and Mnb protein levels ............................................................. 21
Fig. 3.2 Mnb regulates synaptic growth............................................................................ 23
Fig. 3.3 Mnb is required for reliable endocytosis ............................................................. 27
Fig. 3.4-1 Mnb binds to and phosphorylates Synj both in vitro and in vivo ..................... 32
Fig. 3.4-2 mnb mutants display lower levels of phosphorylated Synj .............................. 33
Fig. 3.5-1 Mnb regulates interaction of endocytic proteins and enhances Synj
5’-phosphoinositol phosphatase activity ............................................................. 38
Fig. 3.5-2 Mnb phosphorylation regulates Synj interaction with endocytic proteins ....... 39
Fig. 3.6 Synaptic activity promotes Mnb-dependent Synj phosphorylation and activity
enhancement ........................................................................................................ 42
Fig. 3.7-1 Upregulation of Synj rescues defective endocytosis in mnb
1
.......................... 46
Fig. 3.7-2 Synj overexpression rescues p-Synj level in mnb
1
........................................... 47
Fig. 4 The model of Mnb-mediated phosphorylation of Synj regulating synaptic
morphology and endocytosis. ............................................................................ 511
vii
TABLE OF ABBREVIATIONS
Abbreviation Full Name
AP Alkaline Phosphatase
Dyn Dynamin
Endo Endophilin
Mnb Minibrain
Synj Synaptojanin
Synt Synaptotagmin
viii
ABSTRACT
Protein phosphorylation by kinases plays an important role in regulating synaptic
development and function. During synaptic activity, coordinated phosphorylation and
dephosphorylation of endocytic proteins dynamically regulate the timing and efficacy of
synaptic vesicle recycling. Minibrain kinase (Mnb), also known as the Dual Specificity
tyrosine kinase (Dyrk1A), is a conserved serine/threonine kinase capable of
phosphorylating endocytic proteins such as Synaptojanin (Synj) and Dynamin in vitro,
but whether Mnb/Dyrk1A works as a synaptic kinase that modulates synaptic function in
vivo is not known. Here we describe the characterization of Mnb in the Drosophila
neuromuscular junction. We find Mnb is essential for normal synaptic growth and vesicle
endocytosis. We show Mnb is located in the presynaptic terminals and can phosphorylate
Synj in vivo. Phosphorylation of Synj by Mnb regulates complex endocytic protein
interactions and uniquely enhances synaptojanin activity. Synaptic activity increases Mnb
colocalization with endocytic proteins and triggers Mnb-dependent phosphorylation and
enhancement of Synj activity. Our data identify Mnb as a novel synaptic kinase that
dynamically regulates Synj function to promote activity-dependent facilitation of
synaptic vesicle recycling.
1
CHAPTER 1: INTRODUCTION
1.1 Synaptic endocytosis
Synaptic vesicle recycling is essential for neurons to retrieve the membranes from
nerve terminal and replenish the vesicles that are released by exocytosis. During synaptic
activity, vesicles are fused with synaptic membrane for releasing neurotransmitters. In
order to maintain the pool of vesicles, synaptic vesicles are recycled by retrieving
synaptic membrane through endocytosis mechanism (Heuser and Reese, 1973). Synaptic
endocytosis is important in maintaining normal synaptic function. Defective endocytosis
is an early clinical feature found in numerous neurological diseases, such as Down
syndrome (DS) and Alzheimer’s disease (AD) (Cataldo et al., 2000; Cataldo et al., 2008).
Hence, understanding endocytotic pathways may provide valuable insights into
mechanisms underlying these diseases.
1.2 Study endocytosis in neuromuscular junctions of Drosophila melanogaster
1.2.1 Drosophila melanogaster
Drosophila melanogaster (Fruit fly) is one of the extensively studied models of
endocytosis. Numerous mutants are available for quick and systematic genetic screening
2
for endocytotic proteins. Several endocytotic proteins are found through genetic
screening, such as Dynamin (shibire) (Chen et al., 1991; van der Bliek and Meyerowitz,
1991) and Endophilin (Verstreken et al., 2002). Furthermore, Drosophila loss-of-function
study of genetic mutants using mutagenesis and gene knockdown using RNAi provides
us a powerful tool to characterize the phenotypes, and study the endogenous functions of
these genes in vivo. On the other hand, gain-of-function study using Gal4/UAS system in
Drosophila enables us to model certain neurological diseases caused by gene
overexpression, including Down syndrome. The capability of upregulating and
downregulating a group of genes sharing spatial and temporal expressions or genes
involved in the same biological pathways using Gal4/UAS system also helps us to
understand the protein interaction network of endocytotic pathways. In addition to the
genetic techniques, accessible preparations for both morphological and physiological
measures of larval neuromuscular junction of Drosophila also give us a better way to
characterize endocytosis pathways.
1.2.2 Drosophila neuromuscular junction
Drosophila neuromuscular junction has been widely used for studying endocytosis
pathways. Distinct synaptic shape and structure provide us a good way to characterize
3
synaptic morphology by measuring the size and the number of synaptic boutons. Since
abnormal synaptic morphology is commonly associated with defective synaptic activity
(Schwarz et al., 2005), changes in synaptic morphology can be used as phenotypes for
screening mutants. Furthermore, the accessibility of neuromuscular junction enables us to
measure synaptic activity by stimulating synapses using electrode or KCl solution. It
allows us to observe synaptic activity in real time. We can also characterize synaptic
activity by labeling synaptic vesicles using fluorescent styryl FM dyes, which are
incorporated into synaptic vesicles through endocytosis and released during exocytosis.
Along with different FM dyes and frequencies of electrode stimulation, we are capable of
labeling different pools of synaptic vesicles, reserve pool, readily-releasing pool and
recycling pool, in synapses (Verstreken et al., 2005). The methods above are able to
providing us valuable insights of how endocytotic proteins interact with each other and
how they are regulated in different stages in endocytotic pathways.
1.3 Endocytosis regulation
1.3.1 Synaptic kinases
Protein phosphorylation by kinases plays an important role in regulating endocytosis
activity. During synaptic activity, coordinated phosphorylation and dephosphorylation of
4
endocytotic proteins dynamically regulate the timing and efficacy of synaptic vesicle
recycling. Several synaptic kinases involved in regulating endocytotic proteins have been
identified and characterized in Drosophila, such as Cyclin-dependent kinase 5 (Lee et al.,
2004) and Leucine-rich repeat kinase 2 (Matta et al., 2012). Although in vitro studies
have shown that there are some other synaptic kinases and their substrates have not been
well-characterized yet (Huang et al., 2004; Adayev et al., 2006), whether these kinases
regulate endocytosis in vivo are still unknown. Hence, in this study, we try to characterize
a synaptic kinase, Minibrain (Mnb), and study how the phosphorylation of its substrate
regulates endocytosis activity.
1.3.2 Minibrain kinase
The Minibrain kinase (Mnb), also known as its mammalian homologue, the dual
specificity tyrosine(Y)-phosphorylation-regulated kinase 1A (Dyrk1A), is a functionally
diverse proline-directed serine/threonine kinase. It was first characterized by Tejedor et al.
(1995) showing that Mnb fly mutants have smaller brains, reduced number of neurons
and locomotor defects. Further study done by Fotaki et al. (2002) using
haploinsufficiency mice also showed the same phenotype. In neurons, Dyrk1A/Mnb is
found in the nucleus and cytoplasm, as well as at the synapse (Hammerle et al., 2002;
5
Marti et al., 2003; Wegiel et al., 2004; Arque et al., 2013). Because of its ability to
regulate neurogenesis and neuronal proliferation, much of the research has focused on the
nuclear function of Dyrk1A/Mnb, given its ability to phosphorylate cyclin and other
transcription and splicing factors (Tejedor and Hammerle, 2011). Although in vitro
studies have shown that Dyrk1A/Mnb can phosphorylate several endocytotic proteins
including synaptojanin 1 (Synj) and Dynamin (Huang et al., 2004; Adayev et al., 2006),
the mechanism of how Dyrk1A/Mnb mediated phosphorylation regulates these
endocytotic proteins during synaptic activity in vivo remains unknown.
1.3.3 Synaptojanin
Synaptojanin (Synj) is a phosphoinositol phosphatase that contains an N-terminal
Sac-1 domain, a 5’-phosphoinositol phosphatase domain, and a C-terminal proline rich
domain (PRD). Synj can interact with other endocytotic proteins including Endophilin
(Endo) and Dap160/intersectin via its PRD to facilitate synaptic vesicle endocytosis
(Ringstad et al., 1997; Roos and Kelly, 1998; Harris et al., 2000; Schuske et al., 2003;
Verstreken et al., 2003; Koh et al., 2004; Marie et al., 2004). Studies have also
demonstrated that Synj activity and interaction with Endo can be inhibited by kinases
6
such as Cdk5 and the ephrin receptor (Lee et al., 2004; Irie et al., 2005), underscoring the
possibility that Synj may undergo dynamic regulation in facilitating endocytosis.
1.4 Goals and significance of the research
Although studies have shown that Dyrk1A/Mnb can interact with certain
endocytotic proteins in vitro (Huang et al., 2004; Adayev et al., 2006), the in vivo
substrates of Dyrk1A/Mnb have not yet been characterized. Furthermore, if Dyrk1A/Mnb
is localized in synapses and involved in endocytosis regulation is not known. In this
research, our goal is to characterize the regulation of endocytosis by Mnb-mediated
phosphorylation. We would like to first demonstrate the phenotypes of abnormal synaptic
morphology and defective endocytosis. Then we would identify if Synj is one of the
major downstream substrates of Mnb in vivo, and how Mnb regulates endocytosis
through Synj phosphorylation. Furthermore, we want to see if phosphorylation of Synj by
Mnb is activity-dependent. At last we check if overexpressing Synj is able to rescue
defective endocytosis in mnb
1
background, which has been identified as hypomorphic
mutants. Since Mnb is known to be mutated in autism spectrum disorder, and both Mnb
and Synj are upregulated in Down syndrome, understanding how Mnb-dependent
7
phosphorylation of Synj regulates endocytosis activity will provide valuable insights into
mechanisms underlying some clinical features of these diseases.
8
CHAPTER 2: EXPERIMENTAL PROCEDURES
2.1 Fly Stocks and antibody generation
The following fly lines were used: mnb
1
(Tejedor et al., 1995), mnb
f0137
(abbreviated
mnb
P
in this manuscript; Exelixis collection, Harvard Medical School), Df
(6217;
Bloomington Stock Center, Indiana University), and UAS- PLC δ 1-PH-GFP (Verstreken et
al., 2009). The synj and mnb-F transgene constructs were generated by subcloning the
coding regions of synj or mnb-F into the pINDY6 vector, which contains the HA tag, as
described previously (Chang et al., 2003), and transgenic flies were generated by
standard transformation method (Montell et al., 1985). To drive neuronal expression,
n-synaptobrevin-Gal4 (nSyb-Gal4) was used (Pauli et al., 2008). Affinity purified rabbit
polyclonal antibody for Mnb was generated against Mnb-F amino acids 21-151, and for
Synj was generated against amino acids 361-535 (PrimmBiotech, Inc).
2.2 Immunochemistry
Third instar larvae were dissected and fixed in 4% paraformaldehyde for 25 min and
washed with 0.1% triton X-100 in PBS. Fixed samples were then blocked with 5%
normal goat serum (NGS) in 0.1% triton X-100 in PBS. Primary antibodies used to label
9
dissected larvae were diluted in blocking solution and used as following: rabbit
anti-p-Synj, 1:2000 (Verstreken et al., 2003); guinea-pig anti-Endo(GP69), 1:200
(Verstreken et al., 2002), guinea-pig anti-Dap160, 1:1000 (Koh et al., 2004); rabbit
anti-Synaptotagmin, 1:1000 (Littleton et al., 1993) , rabbit anti-Mnb, 1:400; rabbit
anti-Synj-1, 1:200; mouse anti-Dynamin, 1:200 (BD Transduction Laboratories); Cy3
conjugated anti-HRP, 1:100 (Jackson ImmunoResearch); mouse anti-bruchpilot (NC82),
1:10 and mouse anti-Dlg (4F3), 1:500 (Developmental Studies Hybridoma bank).
Secondary antibodies used were Alexa-488, 555 or 405 conjugated, 1:250 (Invitrogen).
Images were captured using Zeiss LSM5 confocal microscope at 63X. Staining intensities
were calculated by normalizing the fluorescence intensity to outlined bouton area using
AxioVs40 4.8.2.0 or Image J.
2.3 Electrophysiology and FM1-43 dye labeling
FM1-43 dye labeling using third instar larvae was performed as described
previously (Chang and Min, 2009). Images were captured using Zeiss LSM5 confocal
microscope. Fluorescence intensities were calculated using Image J and were normalized
to the average labeling fluorescence intensity in controls. The ratio of unloaded/loaded
was calculated by subtracting the fluorescence intensity remaining after unloading from
10
the intensity of FM1-43 loading, and then divided by loaded fluorescence intensity:
(F
loaded
– F
unload
)/F
loaded
.
Electrophysiology was performed using an Olympus BX51W1 fixed stage
microscope equipped with a 40x 0.8 NA water dipping objective. Third-instar larvae were
dissected and then bathed in a modified HL-3 saline (in mM): NaCl 70, KCl 5, MgCl
2
10,
NaHCO
3
10, sucrose 115, trehelose 5, HEPES 5 (pH 7.2) (Stewart et al., 1994) with 0.5
mM CaCl for spontaneous miniature EPSP recordings or 2 mM CaCl for evoked EPSP
recordings. Current-clamp recordings were performed on muscles 6 and 7 in abdominal
segments A2, A3, or A4, and severed ventral nerves were stimulated with suction
electrodes at 3 ms stimulus duration. A recording electrode (15-30 MΩ resistance) filled
with 3M KCl was used and data was only analyzed from muscles with a resting potential
more hyperpolarized than -60 mV , input resistance of at least 5 MΩ, and resting
potentials that did not deviate by more than 10 mV for the duration of the recording. Data
sets were rejected in which the stimulated nerve did not function throughout the
recording, as determined by abrupt drops in EJP amplitude. Data was acquired using an
Axoclamp 900A amplifier, digitized using a Digidata 1440A, and controlled using
pClamp 10.3 software (Molecular Devices, Sunnyvale, CA). Electrophysiological sweeps
11
were sampled at a rate of 10 kHz and filtered at 400 Hz. Data was analyzed using
MiniAnalysis (Synaptosoft), SigmaPlot (Systat Software), and Microsoft Excel. Average
EJP amplitude was corrected using nonlinear summation (Martin, 1955).
2.4 Western blotting
Drosophila adult head extract was obtained by homogenizing 12-15 adult heads
collected on dry ice in high EDTA RIPA buffer (50 mM Tris-HCl, pH7.5, 1% NP-40,
0.5% NaDoc, 150 mM NaCl, 0.1% SDS, 10 mM EDTA, 50 mM NaF, 1 mM Na
3
VO
4
,
cycloporin A, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail 1
was separated by
SDS-PAGE and transferred to nitrocellulose membranes. Primary antibodies were diluted
in blocking solution as following: rabbit anti-p-Synj (1:5000); guinea pig
anti-Endo(GP60), 1:200; rabbit anti-Dap160, 1:5000; rabbit anti-Mnb, 1:500, rabbit
anti-Synj-1-rabbit. 1:200; mouse anti-Dynamin, 1:200; mouse anti-Complex V , 1:10,000
(Capaldi et al., 2004); rabbit anti-Phosphothreonine, 1:200 and rabbit anti-Phosphoserine,
1:200 (EMD Millipore).
2.5 Immunoprecipitation
Flies were collected and frozen on dry ice and fly heads were isolated by passing
12
through molecular sieves. Head protein extracts were obtained by homogenization as for
Western blotting. 1.5 mg of protein homogenate was incubated with 1-5 μL antibody
against specific proteins at 4°C overnight. Protein complexes were precipitated by
incubating with protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology) at 4°C for
2 h. The beads were washed with PBS four times and protein complexes were eluted by
SDS-PAGE sample buffer.
2.6 Phosphoprotein separation
Isolation of phosphorylated and non-phosphorylated proteins was achieved using the
PhosphoProtein Purification Kit (Qiagen) according to manufacturer’s instructions. To
ensure complete isolation of phospho- and non-phosphorylated proteins, 1.5 mg of
protein homogenate was passed through two PhosphoProtein purification columns per
genotype. Efficiency of phosphoprotein isolation was confirmed via Western blots using
antibodies against phospho-serine and phospho-threonine.
2.7 Protein purification
His tagged Mnb (His-Mnb) and His and HA doubly tagged Synj (His-Synj-HA)
were obtained by cloning the Mnb-F and Synj-HA sequences from the pINDY6 vectors
(used for transgenic fly generation) into pET15b vector (Novagen). BL21(DE3)
13
competent E.coli. strain containing the expression plasmids were grown at 37°C until
A600 of the culture reached 0.6-0.8. Expression of the proteins was induced by the
addition of isopropyl β−D-thiogalactopyranoside (IPTG) to a final concentration of 1.0
mM. After growth at 30 °C for 4 hours, cells were harvested and stored at -80°C until
purification. For Mnb purification, His-Mnb was purified and eluted using the Ni-NTA
Purification System (Invitrogen). Eluted His-Mnb was dialyzed into kinase buffer (20
mM HEPES (pH 7.4), 10 mM MgCl2, 1 mM DTT and 2 mM Na
3
VO
4
) using
Slide-A-Lyzer Dialysis (ThermoScientific). For Synj purification, IPTG induced bacterial
cell lysate was incubated with anti-HA-agarose beads (Sigma Aldrich) at 4°C overnight
and washed with PBS for four times. To purify Synj from flies, adult head extract was
prepared from flies overexpressing Synj-HA in neurons as described for
immunoprecipitation. Extract was then incubated with anti-HA-agarose beads (Sigma
Aldrich) at 4°C overnight and wash with PBS.
2.8 In vitro dephosphorylation and re-phosphrylation of Synj
Purified Synj-coupled to HA agarose beads (0.5 μg) was dephosphorylated by
incubating with 5 units of alkaline phosphatase (CIP) in NEBuffer 3 (New England
BioLabs) at 37°C for 1 h. Dephosphorylated Synj-coupled to HA agarose beads was then
14
washed with PBS four times to remove CIP. For Mnb rephosphorylation, samples were
subsequently treated with purified Mnb (0.5 μg) in kinase buffer (20 mM HEPES, pH 7.4,
10 mM MgCl
2
, 1 mM DTT and 2 mM Na
3
VO
4
) at 37°C for 1 h and then washed with
PBS for four times to remove Mnb.
2.9 Phosphatidylinositol 5’-phosphatase activity in vitro
Phosphotidylinositol 5’-phosphatase activity was determined using either fly head
extracts or purified Synj-coupled to HA agarose beads. Adult fly heads were
homogenized in a buffer containing 10 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM EGTA,
1% NP-40, 1 mM Na
3
VO
4
, 50 mM NaF, 250 nM cycloporin A and protease inhibitor
cocktail (Roche). 1 μg of protein extract was used per assay. Alternatively, 0.5 ug of
purified Synj coupled to HA agarose beads was used. Protein extract or purified Synj was
incubated with labeled PI(4,5)P
2
(GloPIPs BODIPY® FL-PI(4,5)P2, Echelon) in inositol
phosphatase activity assay buffer (30mM HEPES, pH 7.4, 100 mM KCl, 10 mM EGTA
and 2 mM MgCl
2
) at room temperature for 10-15 min or at 37°C for 5-10 min.
BODIPY-FL PI
4
P and BPDIPY-FL PI(4,5)P
2
were also spoted in separate lanes as
standards. Lipid products were separated by TLC and visualized under UV . Densitometry
analyses were done using Image J. Synj was also eluted by adding SDS-PAGE sample
15
buffer and Synj level was determined by Western blots. Phosphotidylinositol
5-phosphatase activity was normalized to the level of Synj.
2.10 In vitro Synj interaction with endocytotic proteins
Wildtype fly head extracts were obtained as for immunoprecipitation and incubated
with 0.5 μg of purified Synj-HA coupled to agarose at 4°C overnight. The protein
complexes were then washed with PBS and eluted in SDS-PAGE sample buffer.
Interactions were analyzed via Western blots using antibodies against specific
endocytotic proteins.
2.11 Synapse activity stimulation by high K
+
For NMJ immunochemistry, third instar larvae were dissected in HL-3 solution
without Ca
2+
(110 mM NaCl, 5 mM KCl, 10 mM NaHCO
3
, 5 mM HEPES, 30 mM
sucrose, 5 mM threalose, 10 mM MgCl
2
, pH 7.2). High K
+
stimulation was achieved by
replacing the solution with 90 mM KCl in HL-3 solution with Ca
2+
(25 mM NaCl, 90
mM KCl, 10 mM NaHCO
3
, 5 mM HEPES, 30 mM sucrose, 5 mM threalose, 10 mM
MgCl
2
, 1.5 mM CaCl
2
, pH 7.2) for 30 s. The samples were immediately fixed in 4%
paraformaldehyde for 25 min and labeled with specific antibodies as for
immunochemistry described previously. Percent increase in p-Synj was calculated by
16
subtracting normalized p-Synj level of unstimulated synapse from normalized stimulated
synapse (p-Synj/Synj-1
stimulate
–p-Synj
unstimulate
/Synj-1
unstimulate
).x100 For in vitro
phosphatidylinositol 5’-phosphatase activity, third instar larvae were stimulated by
incubating in 90 mM KCl in HL-3 solution with Ca
2+
for 5 min and immediately
homogenized in inositol phosphatase activity assay buffer (30mM HEPES (pH 7.4), 100
mM KCl, 10 mM EGTA and 2 mM MgCl
2
). Phosphatidylinositol 5’-phosphatase activity
was determined as described previously. The difference in phosphatidylinositol
5’-phosphatase activity was calculated by subtracting the PI4P/PI(4,5)P
2
ratio of
unstimulated larvae from stimulated larvae.
17
CHAPTER 3: RESULTS
3.1 Mnb is enriched in the presynaptic terminals of Drosophila NMJ
To understand the role of Mnb in synaptic function, we first determined the
localization of the Mnb protein by generating an antibody against all Mnb isoforms (Fig.
3.1-1A). Western blot analyses revealed that Mnb is enriched in the Drosophila nervous
system. Three main bands were detected at approximately 100, 69 and 66 KD, close to
the 96, 66, and 65 KD predicted for Mnb-E, Mnb-F, and Mnb-G, respectively (Fig.
3.1-1B). Mnb-H, which has a predicted molecular weight of 111.1 KD, was below the
level of detection. Specifically, Mnb-F and Mnb-G are enriched in the adult heads and
larval brains, whereas Mnb-E is found in both the body muscle wall and the brain. This
result is consistent with our quantitative PCR results, which showed that Mnb-F is the
predominant isoform found in neurons (Fig. 3.1-2A).
We next used immunocytochemistry to determine if Mnb protein is present at the
NMJ. Fig. 3.1-1C shows that Mnb reactivity is mostly confined to the boundaries of HRP
staining, a marker for the presynaptic membrane (Jan and Jan, 1982), and did not
colocalize with disc-large (Dlg), a postsynaptic maker (Parnas et al., 2001). This result
indicates that Mnb is a novel kinase enriched in the presynaptic boutons of the
18
Drosophila NMJ, which is also consistent with a recent report that demonstrated Dyrk1A
is found in a subset of motor neurons and present in the mouse NMJ (Arque et al., 2013).
To gain insights into the functional aspects of Mnb kinase in the synapse, we further
defined Mnb distribution within the synaptic terminals. We observed partial
colocalization of Mnb with either Endo or Dap160, endocytic proteins found in the
periactive zone (Roos and Kelly, 1999; Guichet et al., 2002; Verstreken et al., 2002; Koh
et al., 2004; Marie et al., 2004) (Fig. 3.1-1D and 3.1-2B). Although punctate, Mnb was
frequently seen adjacent to NC82 staining, an active zone marker (Kittel et al., 2006;
Wagh et al., 2006), we did not detect distinct colocalization. Together, these results
indicate Mnb is found in subdomains of periactive zones and may affect synaptic vesicle
recycling.
Synaptic kinases have been shown to play a major role in sculpting the synapse by
either promoting or restraining synaptic growth (Aberle et al., 2002; Marques et al., 2002;
Collins et al., 2006; Zhang et al., 2007; Kim and Snider, 2011; Hruska and Dalva, 2012).
Mnb is found at the synapse, but it is not known if it plays any role in synaptic
development or morphogenesis. Previous studies in flies and mice have demonstrated that
null mutations in Mnb cause embryonic lethality (Fotaki et al., 2002; Hong et al., 2012).
19
Hence, we studied the effects of Mnb in synaptic growth and function using hypomorphic
alleles. mnb
f0137
, a mutant with a transposon insertion in the 5’-UTR of the mnb gene (Fig.
3.1-1A), was found to show a mild reduction in the level of overall Mnb protein as
assessed by Western blots and specifically in the synapse as indicated by immunostaining
(Figs. 3.1-1E and 3.1-2C). mnb
1
, the classical mnb mutant, which harbors a point
mutation next to the critical ATP binding residue in the kinase active site previously
shown to influence both Mnb function and expression levels (Tejedor et al., 1995), also
showed a corresponding decrease in Mnb levels in the synaptic terminal. To further
reduce Mnb protein, we generated mnb
1
alleles in trans to a mnb deficiency allele
(mnb
1
/Df), which exhibited a ~10% survival rate and displayed the largest reduction in
Mnb levels (Fig. 3.1-1E and Fig. 3.1-2C). Since the mnb-F isoform is the most abundant
isoform found in neurons, we also generated transgenic flies expressing mnb-F using the
UAS/GAL4 system. Figs. 3.1-1E and 3.1-2D show that expression of mnb-F using the
pan-neuronal synaptobrevin-Gal4 driver (syb-Gal4) indeed caused overexpression of
Mnb.
3.2 Mnb is required for synaptic growth
We examined synaptic morphology in different mnb alleles by immunostaining the NMJ
20
Fig. 3.1-1 Mnb is found in the presynaptic terminals of Drosophila NMJ. (A)
Schema of predicted mnb isoforms as described in Flybase. Orange boxes indicate
translated region and gray boxes show untranslated mRNA. Green lines and inverse
triangles indicate mutant alleles. * indicates ATP binding site in the kinase domain
(KD). Blue line indicates region used to generate MNB antibody (Ab). (B)
Representative Western blot showing enrichment of mnb isoforms in the larval brain
and adult head extracts. * indicates a non-specific band. (C) 3
rd
instar NMJ of muscle
6/7 at A2 stained by MNB. Mnb colocalizes with HRP but not Dlg, indicating presence
in the presynaptic terminal. (D) Image of a single bouton stained with Mnb, Endo, and
NC82 to show colocalization of Mnb with subdomains of Endo in the periactive zone.
Mnb did not colocalize with NC82. (E) Levels of Mnb staining within the NMJ of 3
rd
instar larvae of the indicated genotype. n = 6-9 NMJs each. * p < 0.05. All values
indicate mean ± SEM.
21
Fig. 3.1-2 Mnb isoforms and Mnb protein levels. (A) RT-qPCR was performed to
determine the relative transcript levels from fly brain extracts. Primers were designed
against predicted isoforms specific for Mnb-F, G, H, or E/H isoforms. (B) Image of a
single bouton stained with Mnb, Dap160 to show colocalization of Mnb with
subdomains of Dap160 in the periactive zone. (C) and (D) Western blots and
quantification of Mnb levels in adult heads of the indicated genotype. n = 3 independent
experiments. * indicates p < 0.05. All values indicate mean ± SEM.
22
with HRP, a marker of the presynaptic neuronal membrane. Fig. 3.2 revealed that
reducing Mnb levels led to an undergrowth phenotype, with the NMJs showing a
profound decrease in the number of synaptic boutons, but an increase in the size of these
terminals. mnb
1
/Df showed the most striking changes in synaptic morphology. To confirm
that Mnb is responsible for altered synaptic growth, we attempted to restore the level of
Mnb-F in neurons of mnb
1
mutants (Figs. 3.1-1E and 3.2). While overexpression of
Mnb-F in neurons alone using syb-Gal4 driver resulted in the opposite phenotype,
namely an increase in the number of smaller boutons, overexpression of mnb-F in
neurons of mnb
1
mutant was sufficient to restore the number and size of synaptic boutons
back to normal. These results confirm that Mnb is required presynaptically for proper
synaptic morphology and growth.
3.3 Mnb is required for rapid synaptic vesicle recycling
Phosphorylation of synaptic proteins was proposed to be a major mechanism
dynamically regulating the complex protein interactions and enzymatic activities to
maintain stable synaptic transmission despite wide ranges of activity (Greengard et al.,
1993; Slepnev et al., 1998; Cousin et al., 2001; Clayton et al., 2010; Matta et al., 2012).
To determine whether Mnb modulates synaptic transmission, we performed
23
Fig. 3.2 Mnb regulates synaptic growth. (A) Muscle 6/7 NMJ at A2 stained by HRP.
Lower panels show magnified images of the white boxed region. (B) Number of
boutons normalized to muscle surface area (MSA). (C) Average Type Ib bouton area. *
indicates p <0.05. n =6-9 NMJs each. All values are mean ± SEM.
24
electrophysiology at the NMJ. These recordings demonstrate that mnb
1
mutants have
enlarged mEJP amplitude (Figs. 3.3A-B), similar to other endocytic mutants (Zhang et al.,
1998; Verstreken et al., 2002; Koh et al., 2004; Marie et al., 2004; Dickman et al., 2005).
mnb
1
mutants showed normal evoked EJP amplitude (Figs. 3.3C and 3.3D), suggesting
that basal synaptic transmission is not altered at low stimulation frequency, which is also
consistent with other endocytotic mutants. Interestingly, when we stimulated the nerve at
a higher frequency (10 Hz) for a prolonged period (10 minutes), mnb
1
showed
significantly faster rundown than the control (Figs. 3.3E-F). This result suggests that Mnb
promotes efficient synaptic vesicle recycling. Overexpression of mnb-F alone did not
affect basal synaptic transmission and responses to high frequency stimulation as
compared to the control, but significantly rescued mEPSP amplitude and rate of rundown
when mnb-F was overexpressed in neurons of mnb
1
(Figs. 3.3A-F). Together, these data
indicate neuronal Mnb is required for rapid synaptic vesicle recycling.
Colocalization of Mnb with endocytic sub-domains suggests that Mnb is required
for endocytosis. To directly measure the recycling synaptic vesicle pool, we loaded the
synaptic terminals with the lipophilic FM1-43 dye during stimulation, which incorporates
into synaptic vesicles during endocytosis and can subsequently be released during
25
exocytosis. The fluorescence intensity corresponds to the net amount of FM1-43 dye
uptake, a function of both exocytosis and endocytosis. Synaptic vesicles of mnb
1
loaded
with FM1-43 dye during synaptic stimulation with 60 mM of KCl for 5 minutes
displayed a significant decrease in FM1-43 dye loading intensity (Figs. 3.3G and 3.3H),
indicating a defect in synaptic transmission consistent with electrophysiology data
obtained for prolonged stimulation. mnb
P
allele showed a similar decrease in loading
intensity, and mnb
1
/Df showed the most dramatic decrease in FM1-43 dye loading. To
further delineate if this is due to an endocytic or exocytic defect, we stimulated the loaded
boutons again without FM1-43 dye to measure the extent of exocytosis. Figs. 3.3H and
3.3I show that mnb mutants have normal exocytosis since the amount of FM1-43 dye that
unloaded (signal remaining after unloading subtracted from signal from loading)
normalized to the amount of loaded was unaltered, thus indicating Mnb reduction causes
a specific defect in endocytosis.
Interestingly, overexpression of mnb in neurons led to increased FM1-43 dye loading
while the amount of FM1-43 dye removed during unloading protocol was also enhanced
(Figs. 3.3H and 3.3I). Together with electrophysiology data indicating normal basal
synaptic transmission and normal rundown during prolonged high frequency stimulation,
26
the FM1-43 experiments suggest that rates of endocytosis and exocytosis are both
enhanced but balanced when Mnb is upregulated. Restoring Mnb level by overexpressing
Mnb in mnb
1
compensated for the decrease in FM1-43 dye loading, consistent with the
electrophysiology data. Together, these results confirm that Mnb facilitates rapid synaptic
vesicle endocytosis.
3.4 Synaptojanin is a substrate for Mnb
Coordinated phosphorylation and dephosphorylation of endocytic proteins located in
the presynaptic terminal dynamically regulates the timing and efficacy of synaptic vesicle
recycling (Greengard et al., 1993; Slepnev et al., 1998; Cousin et al., 2001; Clayton et al.,
2010; Matta et al., 2012). Dyrk1A/Mnb has been shown to phosphorylate a number of
endocytic proteins including Synj and Dynamin in vitro (Huang et al., 2004; Adayev et
al., 2006), but there is a lack of in vivo evidence and the physiological consequence of
Dyrk1A/Mnb phosphorylation are not well understood. Because Mnb regulates
endocytosis, we tested the possibility that Synj is a potential substrate of Mnb. To this
end, we first determined if Mnb can biochemically interact with Synj by performing an
immunoprecipitation assay. As seen in Fig. 3.4-1A, Mnb-F was found in the pull-down
with Synj, indicating that Mnb can bind to Synj. To further examine the effects of Mnb on
27
Fig. 3.3 Mnb is required for reliable endocytosis. (A) Representative mEPSP. (B)
Average mEPSP amplitude. (C) Representative evoked EPSP and (D) average
evoked EPSP. (E) EPSP recordings during 10 Hz Stimulation for 10 minutes. (F)
Relative EPSP amplitude plotted over time for the indicated genotypes. n >5 for
each genotype. * marks p < 0.05 at the indicated time and beyond compared to
control . (G) Images of NMJs after FM1-43 loading and unloading. (H) FM1-43
loading and unloading intensity. (I) Quantification of FM1-43 signal removed
during unloading normalized to amount of loading. For H and I, n = 6-8 NMJs, *
indicates p < 0.05. All values represent mean ± SEM.
28
Synj phosphorylation, we obtained an antibody against the PRD of fly Synj (Verstreken et
al., 2003). Upon alkaline phosphatase (AP) treatment, we found that fortuitously, this
antibody recognizes only the phosphorylated form, since no signal was detected
following AP treatment using this antibody (Fig. 3.4-1B; p-Synj). Note that the lack of
signal is not due to protein degradation since Synj is still detected using another antibody
generated against part of the SAC-1 domain of Synj (Fig. 3.4-1B; Synj-1). To further
ensure that Synj-1 antibody is specific for Synj, we immunoprecipitated Synj using
Synj-1 and detected the eluate with both the p-Synj and Synj-1 antibodies. Fig. 3.4-1C
confirms that Synj-1 antibody is specific and can detect both the phospho- and
non-phospho-Synj.
We took advantage of these two Synj antibodies and tested if Mnb can directly
phosphorylate Synj. We pre-treated purified Synj expressed in bacteria with AP, which
removed most of the existing phosphorylation, and then incubated Synj with purified
His-Mnb protein. Fig. 3.4-2A shows that addition of purified Mnb significantly increased
p-Synj signal while total Synj level remain unchanged, indicating that Mnb can indeed
directly phosphorylate Synj. Interestingly, we noticed that Synj expressed in bacteria has
Mnb phosphorylation site(s) already saturated, since addition of Mnb without AP
29
treatment failed to increase Synj phosphorylation detected by p-Synj antibody (Figs
3.4-2A and 3.4-2B). This may explain why the p-Synj antibody originally generated
using the PRD of Synj purified from bacteria as immunogen is specific for phospho-Synj.
Addition of purified Mnb to Synj immunocipitated from fly extracts also significantly
increased p-Synj signal with or without prior AP treatment (Fig. 3.4-1D), confirming that
Synj is a substrate of Mnb and further imply dynamic regulation of Synj phosphorylation
by Mnb.
Having demonstrated that Mnb can bind to and phosphorylate Synj, we next
examined if Mnb regulates synaptojanin phosphorylation in the synapse. This was
achieved by immunostaining the Drosophila NMJ with either p-Synj or Synj-1 antibodies.
We found that mnb
1
displayed a significant decrease in the level of phospho-Synj while
the total level of synaptojanin as detected by Synj-1 remains unchanged (Figs. 3.4-1E and
3.4-1F). mnb
1
/Df showed a further decrease in Synj phosphorylation, whereas Mnb
overexpression in neurons caused a significant increase in phospho-Synj level locally
within the synaptic terminals. Upregulating Mnb in mnb
1
mutant restored the decrease in
Synj phosphorylation level (Fig. 3.4-1F), confirming that Mnb regulates Synj
phosphorylation level in vivo. Similar results were obtained via Western blot analyses
30
using antibodies against p-Synj and Synj-1 (Figs. 3.4-2C and 3.4-2D). In addition, a
reduction in Synj phosphorylation levels in mnb
1
was confirmed using phospho-serine
and phospho-threnonine specific antibodies following immunoprecipitation of Synj,
which showed specific reduction in the levels of Synj phosphorylated on serine and
threonine residues (Fig. 3.4-2E). Taken together, our data suggest that Mnb is a novel
synaptic kinase that regulates Synj phosphorylation in the synapse.
We also examined the possibility that Mnb phosphorylates other endocytic proteins to
regulate synaptic vesicle recycling. To this end, we purified or enriched for
phosphorylated proteins from fly heads with phosphoprotein purification columns, which
offers the advantage of simultaneous isolation of proteins phosphorylated on the serine,
threonine, and tyrosine residues (Makrantoni et al., 2005), as well as systematic detection
of the level of different phosphorylated and non-phosphorylated proteins using Western
blot analyses. After confirming that phospho-protein was effectively isolated from the
non-phosphorylated proteins by Western blot analysis with phosphoserine antibody (Fig.
3.4-2F), we next examined the effect of mnb mutation on phosphorylation of Dynamin,
Dap160, and Endo. This was achieved by calculating the fold change in the amount of
respective protein detected in the phosphorylated fraction and normalized to the total
31
level using Western blots. Changes in the level of Synj phosphorylation was also
examined as a control. We find that compared to control, mnb
1
flies showed a lower
proportion of phosphorylated Synj, consistent with our results using the Synj antibody
specific for phospho-Synj as above. However, mnb mutation did not reduce the
phosphorylation of Dynamin, Endo, or Dap160 (Fig. 3.4-2G). Since Dynamin was
previously shown to be a substrate of Dyrk1A/Mnb in vitro (Chen-Hwang et al., 2002;
Huang et al., 2004), we further validated these results by immunoprecipitating Dynamin
from control and mnb
1
flies, and determined the extent of Dynamin phosphorylation
using phospho-serine or phospho-threonine specific antibodies. Figs 3.4-1G and 3.4-1H
show that mnb
1
flies displayed normal levels of Dynamin phosphorylated on the serine
and threonine residues. Thus, Mnb is not likely to influence endocytosis through altered
phosphorylation of Dynamin, Endo, or Dap160, and Synj seems to be the specific
endocytic substrate in vivo.
3.5 Mnb regulates interaction of endocytic proteins and enhances Synj
5’-phosphoinositol phosphatase activity
The PRD at the C-terminus of Synj is capable of binding to multiple endocytic
proteins with SH3 domains such as Endo and Dap160 to coordinate uncoating of synaptic
32
Fig. 3.4-1 Mnb binds to and phosphorylates Synj both in vitro and in vivo. (A)
Immunoprecipitation experiment using flies overexpressing Synj tagged with HA
reveals that Synj interacts with Mnb-F. (B) Alkaline phosphatase (AP) treatment
shows that the p-Synj antibody is specific for phospho-Synj. (C)
Immunoprecipitation experiment done using Synj-1 antibody followed by AP
treatment reveals that Synj-1 can also detect both phosphorylated and
non-phosphorylated Synj. (D) Incubation of immunoprecipitated Synj in the
presence and absence of AP and Mnb pre-treatment. Mnb enhanced Synj
phosphorylation as revealed by p-Synj antibody. Lower graph shows quantification
of relative p-Synj and Synj-1 signals in the indicated treatment conditions. n = 3, *
indicates p < 0.05. (E) Staining of Synj in the 3
rd
instar NMJ done using p-Synj and
Synj-1 antibodies for the indicated genotypes. mnb overexpression increased p-Synj
staining in the synapse while Mnb reduction in mutants decreased the level of
p-Synj. (F) Quantification of relative staining intensity for p-Synj and Synj-1
signals. n = 6-8 NMJs, * indicates p < 0.05. (G) Immunoprecipitation done using
dynamin antibody, and the levels of phospho-serine (p-Ser) and phospho-theronine
(p-Thr) were determined using phospho-specific antibodies as indicated. (H)
Quantification of relative levels of p-Ser and p-Thr after normalizing to the amount
of dynamin in the IP. n = 3, p < 0.05. All values represent mean ± SEM.
33
Fig. 3.4-2 mnb mutants display lower levels of phosphorylated Synj. (A)
Incubation of purified Mnb with Synj purified from bacteria increases the level of
p-Synj only if Synj had been pretreated with alkaline phosphatase (AP). (B) Mnb
does not further enhance p-Synj signal without prior AP treatment. (C) Western blots
of adult fly head extracts detected with p-Synj and Synj-1 antibodies for the
indicated genotypes. (D) Quantification of p-Synj and total Synj levels for the
indicated genotypes. (E) Immunoprecipitation (IP) performed using Synj-1 antibody.
Levels of Synj-1 phosphorylated on serine (p-Ser) and threronine (p-Thr) were
determined using phospho-serine and phosphor-threonine specific antibodies. Values
were normalized to the amount of Synj-1 in the IP. n = 3 experiments. (F) Western
blot using p-Ser antibody demonstrates that phosphorylated proteins were enriched
in “phospho elute” while the non-phosphorylated proteins were enriched in the
“flow through” fractions. (G) The level of phosphorylated proteins was determined
by Western blots using antibodies against specific proteins in the “phospho elute”
and “flow through” fractions. After normalizing to control, mnb
1
has lower level of
phosphorylated Synj while the level of phosphorylated dynamin, Endo and Dap160
did not change. n = 3 independent experiments. * indicates p < 0.05. All values are
mean ± SEM.
34
vesicles during endocytosis (Micheva et al., 1997; Ringstad et al., 1997; Roos and Kelly,
1998; Schuske et al., 2003; Verstreken et al., 2003; Koh et al., 2004; Marie et al., 2004).
Furthermore, it was shown that phosphorylation of Synj within the PRD by kinases such
as Cdk-5 and Ephrin receptor negatively regulates its interaction with Endo (Lee et al.,
2004; Irie et al., 2005), and the effect of Dyrk1A phosphorylation on the interaction
between Synj and Intersectin in vitro was shown to be complex and difficult to interpret
(Adayev et al., 2006). We thus examined the effect of Synj phosphorylation by Mnb on
Synj recruitment and interaction. Pull down studies using Synj-HA expressed in bacteria
revealed that Mnb incubation (following pre-treatment with AP) not only increased the
level of Synj phosphorylation, but also reduced Synj interaction with Endo while
increasing Dap160 interaction (Figs. 3.5-2A and 3.5-2B). This result suggests that
Mnb-dependent phosphorylation of Synj shifts its interaction preference to Dap160. To
further establish Mnb functions in vivo, we examined Synj interaction using
immunoprecipitation studies. Figs. 3.5-1A and 3.5-1B show that mnb
1
mutants exhibited
increased Synj-Endo interaction but reduced Dap160 binding as compared to the control,
indicating that Mnb alters the endocytic scaffold complex and may act to regulate
recruitment of Synj during clathrin-mediated endocytosis. To confirm that this decrease
35
in Synj-Dap160 is not due to altered Dap160 level, which is an important scaffolding
protein required for proper localization of endocytic proteins, we also immunostained the
NMJs with Dap160 antibody in control, mnb
1
and mnb
1
/Df lines (Fig. 3.5-2C-3.5-2D).
Note that no change was observed, and the levels of Endo and Syt are also normal (Fig.
3.5-2C-3.5-2D).
Since the PRD of either Synj or Dynamin binds to a single SH3 domain of
Endophilin (Ringstad et al., 1997; Shupliakov et al., 1997; Verstreken et al., 2002;
Verstreken et al., 2003), we further examined the possibility that altered Synj
phosphorylation indirectly influences the proper interaction between Dynamin and
Endophilin in vivo. Interestingly, mnb
1
mutants showed reduced interaction between
Endo and Dynamin (Figs. 3.5-1C and 3.5-1D). This result was further confirmed by
immunoprecipitation performed using Endo antibody (Figs. 3.5-1E and 3.5-1F), which
confirmed reduced interaction between Endo and Dynamin, but enhanced Endo-Synj-1
interaction in mnb
1
as compared to control. Collectively, these data demonstrate that
Mnb-mediated Synj phosphorylation modulates the precise interaction between multiple
endocytic partners, perhaps ensuring robust and rapid endocytosis, particularly during
time of high activity.
36
Synj is a phosphoinositol phosphatase capable of regulating PI(4,5)P
2
levels, which
modulates endocytosis and synaptic growth (Harris et al., 2000; Verstreken et al., 2003;
Di Paolo et al., 2004; Lee et al., 2004; Dickman et al., 2005; Mani et al., 2007; V oronov
et al., 2008; Khuong et al., 2010). We thus next tested the hypothesis that phosphorylation
of Synj by Mnb alters Synj phosphoinositol phosphatase activity. Addition of purified
Mnb, which has been shown to directly phosphorylate Synj, increased the 5’-phosphatase
activity of immunoprecipitated Synj in vitro as assayed by increased conversion of
BODIPY-labeled PI(4,5)P
2
to BODIPY-PI(4)P using TLC (Figs. 3.5-1G and 3.5-1H)
(Chang and Min, 2009). This finding indicates that Mnb-mediated phosphorylation of
Synj directly enhances Synj activity.
To further confirm that Mnb phosphorylation indeed influences synaptojanin activity
in vivo amid complex kinase and phosphatase signaling, we examined Synj activity in
mnb mutants using two complementary approaches. First, phosphoinositol
5’-phosphatase activity was determined from control and mnb mutant extracts using TLC.
Figs. 3.5-2E and 3.5-2F show that mnb
1
and mnb
1
/Df flies showed lower level of
converted PI(4,5)P
2
, confirming that the overall Synj activity is reduced in flies with
lower Mnb expression levels. This decrease in Synj activity parallels reduced Synj
37
phosphorylation level (Figs. 3.4-2C and 3.4-2D), consistent with enhancement of Synj
activity due to Mnb. Second, to study if Mnb regulates Synj activity locally at the
synapse, we used the EGFP fusion protein containing the phospholipase Cδ1 pleckstrin
homology domain (PLCδ1-PH-GFP) (Varnai and Balla, 1998; Verstreken et al., 2009).
PLCδ1-PH-GFP has been shown to specifically bind to PI(4,5)P
2
, and the fluorescence
intensity in the synapse directly represents the level of PI(4,5)P
2
. NMJs were labeled with
HRP to outline synaptic boutons and the intensity of the PLCδ1-PH-GFP signal was
examined for different mnb mutants. Consistent with our hypothesis, we find higher
PLCδ1-PH-GFP signal in mnb
1
and mnb
1
/Df NMJs, implying lower Synj activity locally
within the NMJ (Fig. 3.5-1I and Fig. 3.5-1J). Overexpression of Mnb-F decreased
PLCδ1-PH-GFP signal in the synaptic boutons, suggesting Mnb upregulation leads to
elevated Synj activity. Upregulating Mnb-F in mnb
1
restored PLCδ1-PH-GFP signal to
normal, confirming that Mnb is responsible for changes in Synj activity. Taken together,
our results suggest that Mnb phosphorylation of Synj enhances its phosphoinositol
phosphatase activity and is required for normal PI(4,5)P
2
in the synapse.
3.6 Mnb phosphorylates and enhances synaptojanin during synaptic activity
Having demonstrated that Mnb can phosphorylate and regulate Synj, we next asked
38
Fig. 3.5-1 Mnb regulates interaction of endocytic proteins and enhances Synj
5’-phosphoinositol phosphatase activity. (A) Immunoprecipitation (IP) performed
using Synj-1 antibody, (C) IP performed using dynamin antibody, and (E) IP done
using Endo antibody followed by Western blots of the IP eluates done using the
indicated antibodies. (B), (D), and (F) quantification of protein levels detected in the
mnb
1
IP eluate as compared control. n = 3. (G) TLC showing conversion of
BODIPY-PIP2 to BODIPY-PIP by Synj-HA immunoprecipitated from fly extracts
with and without addition of purified Mnb protein. Lower panels show levels of
phospho-Synj and total Synj as detected by p-Synj and Synj-1 antibodies, respectively.
(H) Quantification of relative PIP to PIP
2
level. n = 4 experiments. (I) PIP2 levels in
the NMJ as measured by PLCδ1-PH-GFP for the indicated genotypes. (J)
Quantification of PIP
2
levels in the synapse. Lower Synj activity results in reduced
conversion of PIP
2
to PIP, and will hence have higher PIP
2
levels detected by
PLCδ1-PH-GFP in the synapse. N = 6-8 NMJs. * indicates p < 0.05 for (B), (D), (F),
(H), and (J). All values are mean ± SEM.
39
Fig. 3.5-2 Mnb phosphorylation regulates Synj interaction with endocytic
proteins. (A) Purified Synj pre-treated with AP was incubated with or without
purified Mnb, followed by incubation with wildtype head fly extracts. Western blots
were then performed to determine interactions between Synj and other endocytic
proteins as indicated. (B) Quantification of endocytic protein levels using the
indicated antibodies. (C) Images of NMJs stained with the indicated antibodies.
(D) Quantification of the level of proteins in the synapse, indicating equal levels of
Dap160, Endo and synaptotagmin (Syt) in control and mnb mutants. n = 6-8 NMJs.
(E) TLC showing conversion of BODIPY-PIP2 to BODIPY-PIP by fly extract for
the indicated genotypes. (F) Quantification of PI
4
P/PIP
2
levels for the indicated
genotypes. n = 3 experiments. All values for (B), (D) and (F) are mean ± SEM. *
indicated p < 0.05.
40
whether Mnb acts during synaptic stimulation to regulate Synj phosphorylation and
activity. To this end, we first determined Mnb localization within the synapse at rest and
after synaptic stimulation induced by high KCl (Fig. 3.6A). Compared to unstimulated
synapses, colocalization between Mnb and Endo increased during synaptic activity, while
colocalization with NC82 was unchanged (Fig. 3.6B). This result suggests that synaptic
activity further enhances redistribution of Mnb to the periactive zone. Next, we examined
if Mnb redistribution influences Mnb-mediated phosphorylation of Synj by
immunocytochemistry using antibodies against p-Synj and Synj-1 (Fig. 3.6C). While
Synj-1 levels in control were the same at rest and following synaptic activity, synaptic
activity increased the level of p-Synj in the synapse (Fig. 3.6C). This result demonstrates
that Synj phosphorylation levels are enhanced during periods of high synaptic activity. To
determine if Mnb is responsible for this activity-dependent increase in Synj
phosphorylation, we compared the percent increase in p-Synj level after stimulation
between control, mnb
1
and mnb
1
/Df. Figs. 3.6C and 3.6D show that mnb
1
and mnb
1
/Df
displayed significantly lower level of increase in p-Synj level compared to control,
confirming that Mnb is required for complete Synj phosphorylation during synaptic
activity.
41
We next tested if Mnb-mediated phosphorylation of Synj influences Synj
5’-phosphoinositol phosphatase activity during synaptic stimulation. Consistent with our
finding that phosphorylation enhances Synj activity, larval extracts isolated from control
before and after stimulation showed a higher level of PI(4,5)P
2
conversion after
stimulation (Fig. 3.6E), indicating synaptic stimulation augments Synj activity. However,
mnb
1
and mnb
1
/Df showed little or no enhancement in Synj activity after stimulation,
suggesting that Mnb critically modulates Synj activity during synaptic stimulation (Fig.
3.6F). Interestingly, although mnb
1
/Df
showed almost no Synj activity enhancement as
compared to mnb
1
(Fig. 3.6F), the level of p-Synj was comparable between the two
genotypes (Fig. 3.6D). This implies that Synj is likely phosphorylated at multiple sites,
but the p-Synj antibody may not distinguish all the sites. Altogether, our results suggest
that Mnb is required for activity-dependent phosphorylation and enhancement of Synj.
3.7 Overexpression of Synaptojanin restores synaptic bouton size and endocytic
function in mnb
1
mutants
Based on findings that mnb
1
displayed reduced Synj activity, we hypothesized that
increasing Synj expression may elevate Synj activity and therefore rescue mnb
1
phenotypes. We first confirmed the levels of phospho-Synj and total Synj level in flies
42
Fig. 3.6 Synaptic activity promotes Mnb-dependent Synj phosphorylation and
activity enhancement. (A) Images of synaptic bouton triple-labeled for Endo, Mnb
and NC82 at rest and after stimulation with high K
+
for 30 s. (B) Pearson’s
correlation coefficient values showing colocalization between Mnb/Endo but not
Mnb/NC82. Stimulation enhanced colocalization between Mnb and Endo. n =5.
*indicates p <0.05 compared to unstimulated bouton.. (C) Images of NMJs stained
with p-Synj or Synj-1 antibodies at rest (unstim) and stimulated (stim) with high K
+
.
(D) Quantification of the increase in p-Synj level after stimulation, normalized to
total Synj level. Compared to control, mnb
1
and mnb
1
/Df show a lower level of
increase in phospho-Synj after stimulation. n = 6-8 NMJs. * indicates p < 0.05. (E)
TLC showing conversion of BODIPY-PIP2 to BODIPY-PIP by larval extract with
and without stimulation with high K
+
. (F) Quantification of the increase in PIP
2
conversion after stimulation. n = 3 independent experiments. * indicates p < 0.05. All
values represent mean ± SEM.
43
overexpressing synj alone (synj OE) or Synj in mnb
1
background (mnb
1
; synj OE; Fig.
3.7-2A-3.7-2B). Overexpression of Synj dramatically increased the level of phospho-Synj,
supporting earlier suggestions that Synj is a phospho-protein (Cousin et al., 2001).
Overexpression of Synj in mnb
1
background reduced the level of phospho-Synj compared
to synj OE alone, albeit the level of phosphorylated Synj is still elevated, likely due to
residual Mnb function in the hypomorphic allele. This confirms that Mnb phosphorylates
Synj in vivo. In addition, we measured relative PI(4,5)P
2
levels within the NMJ using
PLCδ1-PH-GFP as an in vivo assay for Synj activity (Figs. 3.7-1A and 3.7-1B).
Consistent with the level of phosphorylated Synj, we found that mnb
1
; synj
OE
significantly rescued the mnb
1
phenotype in terms of PI(4,5)P
2
content. A plausible
explanation is that overexpression of Synj inevitably enhances its activity even in the
presence of reduced levels of Mnb. Immunostaining of the NMJ with HRP revealed that
Synj upregulation in mnb
1
restored the bouton size, but not the number of boutons (Figs.
3.7-2C-3.7-2E), indicating that Mnb-dependent modulation of Synj activity regulates size,
and not the number of boutons. This result is further supported by experiments in which
overexpression of PLCδ1-PH-GFP, which was also used to locally deplete PI(4,5)P
2
(Holz et al., 2000; Raucher et al., 2000; Field et al., 2005; Khuong et al., 2010),
44
effectively restored bouton size but did not alter the number of boutons (Figs. 3.7-2F and
3.7-2G).
Our previous results have shown that overexpression of synj alone does not
influence synaptic vesicle recycling (Chang and Min, 2009), we thus next tested the
ability of synj overexpression to restore the endocytic defect seen in mnb
1
mutants. We
find that synj overexpression in mnb
1
background significantly rescued the faster
rundown phenotype seen in mnb
1
during stimulation (Figs. 3.7-1C and 3.7-1D). However,
after prolonged stimulation at 10 Hz (after 450 s of stimulation), we noticed that NMJs
with synj overexpression in mnb
1
background also began to display a faster rundown than
the control. This is likely attributed to inefficient phosphorylation and enhancement of
Synj activity during synaptic stimulation in mnb
1
background, despite the increase in Synj
protein level. FM1-43 dye labeling experiments further confirmed that Synj
overexpression rescued altered endocytosis in mnb
1
mutant (Figs. 3.7-1E-3.7-1G).
Interestingly, although synj overexpression significantly rescued the synaptic phenotypes
seen in mnb
1
, it did not restore the decrease in brain size seen in mnb
1
(Figs. 3.7-2H and
3.7-2I). These results suggest that Mnb has multiple roles in the nervous system and that
it likely acts locally within the synapse to modulate synaptic vesicle endocytosis.
45
Together, our data demonstrate that Synj is a major substrate of Mnb and requires Mnb
phosphorylation to facilitate rapid endocytosis during periods of high synaptic activity.
46
Fig. 3.7-1 Upregulation of Synj rescues defective endocytosis in mnb
1
. (A)
Representative images of PIP
2
levels in the NMJ as measured by PLCδ1-PH-EGFP
for the indicated genotypes. (B) Quantification of PIP
2
levels. Synj overexpression
in mnb
1
has lower PI(4,5)P
2
level than mnb
1
in the synapse, suggesting a rescue of
Synj activity. n = 6-8 NMJs. * indicates p < 0.05 compared to control and as
indicated. (C) Representative EPSP traces during 10 Hz stimulation for the indicated
genotypes. (D) Relative EPSP amplitude plotted over time for the indicated
genotypes. n = 6 and * indicates p < 0.05 from the indicated time point and beyond
as compared to control. ** p < 0.05 as compared to mnb
1
. (E) Images of NMJ after
FM1-43 loading and unloading. (F) Relative FM1-43 loading and unloading
intensity normalized to control. (G) Quantification of FM1-43 signal removed
during unloading normalized to amount of loading. For (F) and (G), n = 6-8 NMJs,
* p < 0.05. All values represent mean ± SEM.
47
Fig. 3.7-2 Synj overexpression rescues p-Synj level in mnb
1
. (A) Staining of Synj
in the 3rd instar NMJ done using p-Synj and Synj-1 antibodies for the indicated
genotypes. (B) Quantification of relative staining intensity for p-Synj and Synj-1
signals. Synj overexpression increases Synj-1 and p-Synj level in mnb
1
synapse. n =
6-8 NMJs, * indicates p < 0.05. (C) Images of NMJ at muscle 6/7, A2, stained with
HRP. Lower panels show magnified images in the white boxed area. (D) and (E)
Quantification of bouton number normalized to muscle surface area (MSA) and
average bouton area, respectively. * indicates P < 0.05. n = 8 NMJs. (F)
Quantification of average type Ib bouton area, and (G) number of boutons normalized
to muscle surface area for indicated genotypes. Depletion of PI(4,5)P
2
by
PLCδ1-PH-EGFP in mnb
1
rescues the bouton size, but not the number of boutons. n =
6-8 NMJs. * indicates p < 0.05. (E) 3
rd
instar larval brains for the indicated genotypes.
(H) Representative images of 3
rd
instar larval brain hemispheres. (I) Quantification of
brain area for the indicated genotypes. Synj overexpression in mnb
1
fails to rescue the
size of brains. n = 6 larval brains. * indicates p < 0.05. All values are mean ± SEM.
48
CHAPTER 4: DISCUSSIONS AND CONCLUSIONS
4.1 Mnb-mediated phosphorylation enhances Synj phosphatase activity
We demonstrate that Mnb is a novel synaptic kinase essential for normal synaptic
morphology and synaptic vesicle endocytosis. We find that Mnb regulates synaptic
development and endocytosis through Synj. Furthermore, our data revealed a new role for
Dyrk1A/Mnb in the synapse, both in regulating Synj 5’-phosphoinositol phosphatase
activity and in coordinating interactions between Synj and other endocytic proteins. Note
that unlike Cdk5 and ephrin receptor kinase currently known to phosphorylate Synj-1
(Lee et al., 2004; Irie et al., 2005), the effect of Mnb phosphorylation on Synj activity is
stimulatory rather than inhibitory. This result is novel and suggests that phosphorylation
of Synj-1 can dynamically regulate its function, and that Mnb exerts additional regulation
on Synj in vivo to maintain rapid synaptic vesicle recycling (Fig. 3.7-2J). Our
observations of increased colocalization of Mnb with endocytic markers and elevated
levels of phosphorylated Synj during synaptic activity further support the claim that Mnb
acts during neuronal stimulation to enhance Synj function in order to sustain
neurotransmission during periods of high demand (Fig. 4).
4.2 Mnb-mediated phosphorylation of Synj regulates proper endocytic protein
49
interactions and recruitments during synaptic activity
Current models for synaptic vesicle endocytosis suggest that intricately coordinated
interaction between distinct endocytic proteins occur during different steps of endocytosis,
and their proper recruitment is essential for normal synaptic vesicle recycling (Saheki and
De Camilli, 2012) . Although the precise timing of biochemical interactions during
different steps is not well understood, it was suggested that Dap160/intersectin is an
important scaffolding protein that maintains Dynamin, Synj, and Endo at the periactive
zone (Roos and Kelly, 1998; Broadie, 2004; Koh et al., 2004; Marie et al., 2004), that
Endo-dynamin interaction facilitates localization of dynamin at the neck of the clathrin
coated pits prior to fission during early step endocytosis (Sundborger et al., 2011), and
that recruitment of Synj by endophilin promotes uncoating of clathrin during late
endocytosis (Schuske et al., 2003; Verstreken et al., 2003; Dickman et al., 2005;
Milosevic et al., 2011). Our data demonstrate that mnb mutants did not affect the overall
levels of Dap160, Synj, and Endo at the NMJ, but shifted the binding preference of Synj
to enhance interactions with Endo with concomitant reductions in Dap160-Synj and
Endo-Dynamin interactions. Thus, Mnb likely organizes biochemical interactions during
synaptic vesicle endocytosis, leading to a shift toward rapid rates during conditions of
50
high activity (Fig. 4).
4.3 Mnb regulates synaptic morphology through Synj phosphorylation and other
pathways
In addition to regulating endocytosis, we found Mnb also influences synaptic
morphology. The increase in synaptic bouton size seen in mnb mutants may be due to its
inability to retrieve membrane following exocytosis and/or through altered Synj activity,
which governs PI(4,5)P
2
availability in the NMJ. PI(4,5)P
2
has been shown to restrict
synaptic growth through activation of presynaptic Wiscott-Aldrich syndrome
protein/WASP (WSP), which is thought to stimulate actin branching to promote synaptic
bouton growth while restricting synapse extension at the NMJ (Mullins and Machesky,
2000; Sechi and Wehland, 2000; Collins and DiAntonio, 2004; Coyle et al., 2004; Rodal
et al., 2008; Khuong et al., 2010). In support of the hypothesis that Mnb partially affects
synaptic growth through Synj and subsequent PI(4,5)P2 level, overexpression of Synj in
mnb
1
background restored bouton size and that expression of PLCδ1-PH-GFP, which had
previously been shown to deplete PI(4,5)P
2
, rescued the change in bouton size in mnb
1
.
Because reducing Synj phosphorylation and activity in mnb
1
is not physiologically
equivalent to an actual loss of Synj protein, the synaptic morphology of mnb
1
was
51
Fig. 4 The model of Mnb-mediated phosphorylation of Synj regulating synaptic
morphology and endocytosis.
52
different from synj null mutants (reported to show either hyperbudding or no change in
synaptic morphology) (Verstreken et al., 2003; Dickman et al., 2006). Note that Synj
overexpression in mnb
1
did not rescue the number of synaptic boutons to normal,
implying that Mnb also affects other pathways independently from Synj to influence
synaptic morphology.
4.4 Future directions
Mnb is a proline-directed kinase with consensus target sequences RPX(S/T)P or
RX(S/T)P (Himpel et al., 2000; Campbell and Proud, 2002). Similar to many of the in
vivo substrates identified to be phosphorylated by Dyrk1A, fly Synj does not contain the
consensus sequence for Dyrk1A/Mnb phosphorylation. Fly Synj does however contain 12
(S/T)P sites, and with 11 of them in the PRD recognized by the p-Synj antibody. Our data
reveal that Mnb reduction decreased the levels of Synj phosphorylated both on the serine
and threonine residues, indicating that Synj is phosphorylated on multiple sites,
consistent with a previous report in vitro (Adayev et al., 2006). The activity-dependent
phosphorylation and enhancement of Synj function by Mnb may provide a mechanism
that tune the speed of synaptic vesicle recycling to meet the demand of the synapse
during periods of high activity. It will be particularly interesting to determine in the future
53
factors regulating Mnb in the synapse, as well as elucidate the dynamic biochemical
interactions that optimize the speed of vesicle recycling over a range of synaptic activity.
As Dyrk1A/Mnb is mutated in Autism spectrum disorder and upregulated in Down
syndrome (Guimera et al., 1996; O'Roak et al., 2012), an understanding of Dyrk1A/Mnb
in regulating synaptic transmission may not only provide valuable information on
fundamental mechanisms regulating endocytosis, but also shed light on cellular
mechanisms leading to dysfunctional synapses in these neurological disorders.
54
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Abstract (if available)
Abstract
Protein phosphorylation by kinases plays an important role in regulating synaptic development and function. During synaptic activity, coordinated phosphorylation and dephosphorylation of endocytic proteins dynamically regulate the timing and efficacy of synaptic vesicle recycling. Minibrain kinase (Mnb), also known as the Dual Specificity tyrosine kinase (Dyrk1A), is a conserved serine/threonine kinase capable of phosphorylating endocytic proteins such as Synaptojanin (Synj) and Dynamin in vitro, but whether Mnb/Dyrk1A works as a synaptic kinase that modulates synaptic function in vivo is not known. Here we describe the characterization of Mnb in the Drosophila neuromuscular junction. We find Mnb is essential for normal synaptic growth and vesicle endocytosis. We show Mnb is located in the presynaptic terminals and can phosphorylate Synj in vivo. Phosphorylation of Synj by Mnb regulates complex endocytic protein interactions and uniquely enhances synaptojanin activity. Synaptic activity increases Mnb colocalization with endocytic proteins and triggers Mnb-dependent phosphorylation and enhancement of Synj activity. Our data identify Mnb as a novel synaptic kinase that dynamically regulates Synj function to promote activity-dependent facilitation of synaptic vesicle recycling.
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Creator
Chen, Chun-Kan
(author)
Core Title
Minibrain kinase enhances synaptojanin activity to facilitate endocytosis during synaptic activity
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Publication Date
07/10/2015
Defense Date
06/04/2013
Publisher
University of Southern California
(original),
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Tag
endocytosis,kinase,OAI-PMH Harvest,synaptic transmission
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English
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Electronically uploaded by the author
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Tokes, Zoltan A. (
committee chair
), Chang, Karen T. (
committee member
), Lu, Wange (
committee member
)
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chunkan@usc.edu,jameshellohi@gmail.com
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Tags
endocytosis
kinase
synaptic transmission